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【文献】Cas9激活p53途径并富集p53突变个体

标签:发布时间:2024-01-22 11:18:56

Cas9通常被引入细胞系以实现CRISPR–Cas9介导的基因组编辑。作者研究了Cas9表达对细胞的基因组和转录活动遗传和转录活动产生的影响。165对人类癌细胞系及其Cas9表达衍生物的基因表达谱显示,Cas9导入后p53途径的上调,特别是在野生型TP53(TP53-WT)细胞系中。这在mRNA和蛋白质水平上得到了证实。此外,在表达Cas9的细胞系中观察到DNA修复水平升高。42个细胞系对的遗传特征表明Cas9的引入可导致p53失活突变的出现和扩增。在同基因TP53-WT和TP53-null(TP53/)细胞系中进行的竞争实验证实了这一点。最后,Cas9在TP53-WT中的活性低于在TP53-突变细胞系中的活性,Cas9诱导的P53通路激活影响细胞对遗传和化学扰动的敏感性。这些发现可能对CRISPR–Cas9介导的基因组编辑的正确使用具有广泛的意义。

Cas9 is commonly introduced into cell lines to enable CRISPR–Cas9-mediated genome editing. Here, we studied the genetic and transcriptional consequences of Cas9 expression itself. Gene expression profiling of 165 pairs of human cancer cell lines and their Cas9-expressing derivatives revealed upregulation of the p53 pathway upon introduction of Cas9, specifically in wild-type TP53 (TP53-WT) cell lines. This was confirmed at the messenger RNA and protein levels. Moreover, elevated levels of DNA repair were observed in Cas9-expressing cell lines. Genetic characterization of 42 cell line pairs showed that introduction of Cas9 can lead to the emergence and expansion of p53-inactivating mutations. This was confirmed by competition experiments in isogenic TP53-WT and TP53-null (TP53−/−) cell lines. Lastly, Cas9 was less active in TP53-WT than in TP53-mutant cell lines, and Cas9-induced p53 pathway activation affected cellular sensitivity to both genetic and chemical perturbations. These findings may have broad implications for the proper use of CRISPR–Cas9-mediated genome editing.


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